Continual addition of posterior tail segments beyond the embryonic period is a little known and little understood phenomenon. The immediate goal of this research is to investigate tail elongation and regeneration across life stages (embryo, larval, and adult) in a non-model salamander species, Eurycea cirrigera (Plethodontidae), which continuously adds vertebrae throughout its life cycle. The central hypothesis of the proposed research is that the ability to continuously add tail segments post-embryonically is functionally related to the ability to regenerate the tail. Postembryonic addition of segments suggests that the tail tip retains at least some aspects of embryonic cell/tissue organization and gene expression throughout the life cycle. Knowledge of how the spinal cord and other tissues continue to develop, and which, if any, Hox genes are expressed in the continually elongating tail tip could reveal important mechanisms that underlie the process of regeneration. Overall, important insights to differentiation, proliferation, and stem cell origin and plasticity may be gained from this work. In order to investigate the hypothesis, the goals of this study are to: 1. Describe morphologically the cellular development and regeneration of the posterior tail. 2. Investigate somitogenesis (segmentation) and expression of select early axial tissue markers (type II collagen and Pax7) across life stages in a non-model salamander that exhibits continual elongation of the tail. 3. Characterize the expression of posterior Hox genes across life stages in the normal and regenerating tail. E. cirrigera embryos will be collected in the field and maintained in laboratory environmental chambers. Standard techniques such as clearing and staining, histology, as well as antibody staining will be used to visualize axial segmentation and vertebral number, and to describe development of caudal tissues such as the spinal cord. The investigators'preliminary genomic PCR screen yielded sequences related to the HoxB9, HoxA10, HoxB13, and HoxC13 paralogs. This screen will be continued and extended to target additional posterior Hox paralogs. Sequence information from cloned homeodomains will be used to design gene specific primers to isolate full-length cDNA clones. RNA will be isolated from embryonic tail buds to generate in situ probes for documenting the expression of select Hox genes across life stages. PUBLIC HEALTH RELEVANCE: This research proposes to investigate how development of the posterior body axis can persist post-embryonically in an organism that also has the ability to regenerate. Understanding how caudal tissues, such as the spinal cord, are re-structured, and identifying which Hox genes are expressed, and how they are expressed will shed light on mechanisms that link continual axial elongation with regeneration. Such a study is important in the ongoing search for mechanisms and factors that could contribute to therapies attempting to induce re-growth of tissue (e.g., nervous tissue) in organisms that have lost the natural ability to regenerate.